New reverse genetics and transfection methods to rescue arboviruses in mosquito cells

Atieh, Thérèse; Nougairède, Antoine; Klitting, Raphaëlle; Aubry, Fabien; Failloux, Anna‐Bella; Lamballerie, Xavier de; Priet, Stéphane

doi:10.1038/s41598-017-14522-6

Cited by 25 publications

(27 citation statements)

References 29 publications

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“…To recover ZIKV variants we used infectious subgenomic amplicons (ISA) (31-33) as previously described (32). For the WT ZIKV variant, three pUC57 plasmids [EVAg 001N-01891] containing overlapping regions (PF-I: 1-3428 nt, PF-II: 3354-7621 nt, PF-III: 7553-10807; all nucleotide (nt) positions based on complete viral strain sequence) covering the full ZIKV H/PF/2013 genome were used (31).…”

Section: Recovery Of Cpg-recoded Zikv Variantsmentioning

confidence: 99%

CpG-Recoding in Zika Virus Genome Causes Host-Age-Dependent Attenuation of Infection With Protection Against Lethal Heterologous Challenge in Mice

et al. 2020

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Experimental increase of CpG dinucleotides in an RNA virus genome impairs infection providing a promising approach for vaccine development. While CpG recoding is an emerging and promising vaccine approach, little is known about infection phenotypes caused by recoded viruses in vivo. For example, infection phenotypes, immunogenicity, and protective efficacy induced by CpG-recoded viruses in different age groups were not studied yet. This is important, because attenuation of infection phenotypes caused by recoded viruses may depend on the population-based expression of cellular components targeting viral CpG dinucleotides. In the present study, we generated several Zika virus (ZIKV) variants with the increasing CpG content and compared infection in neonatal and adult mice. Increasing the CpG content caused host-age-dependent attenuation of infection with considerable attenuation in neonates and high attenuation in adults. Expression of the zinc-finger antiviral protein (ZAP)-the host protein targeting viral CpG dinucleotides-was also age-dependent. Similar to the wild-type virus, ZIKV variants with the increased CpG content evoked robust cellular and humoral immune responses and protection against lethal challenge. Collectively, the host age should be accounted for in future studies on mechanisms targeting viral CpG dinucleotides, development of safe dinucleotide recoding strategies, and applications of CpG-recoded vaccines.

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Section: Recovery Of Cpg-recoded Zikv Variantsmentioning

confidence: 99%

CpG-Recoding in Zika Virus Genome Causes Host-Age-Dependent Attenuation of Infection With Protection Against Lethal Heterologous Challenge in Mice

et al. 2020

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“…The ISA reverse genetics method had been used in the past for a variety of viruses with a positivestranded RNA genome such as aviviruses, alphaviruses or enteroviruses [14][15][16][17][18] . The method was proven to be extremely simple, rapid and easy to implement, but all previous results were obtained with "short" genomes (i.e.…”

Section: Discussionmentioning

confidence: 99%

“…An obvious consequence of this mechanism of action is that the probability for an individual cell to receive simultaneously upon transfection all subgenomic fragments decreases when the number of fragments increases. We previously showed that for some small (+) ssRNA viruses such as enteroviruses, the number of fragments used can be balanced by increasing in parallel the total amount of DNA to be transfected 15 . However, in the case of coronaviruses, given the length of the genome, this requirement had to be met with amounts of DNA that do not jeopardize cell biological functions, growth, and subsequent replication of the virus.…”

Section: Discussionmentioning

confidence: 99%

“…The Infectious Subgenomic Amplicons (ISA) method is a simple and rapid bacterium-free method that has been developed in recent years and allowed rescuing viruses with relatively small (+) ssRNA genomes, from the Flaviviridae, Togaviridae and Picornaviridae families [14][15][16][17][18] . Unlike conventional reverse genetics systems that require cloning steps, the ISA method is based on the simple transfection of overlapping subgenomic DNA fragments, covering the entire virus genome into permissive cells.…”

Section: Introductionmentioning

confidence: 99%

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Infectious Subgenomic Amplicons method to expedite reverse genetics of SARS-CoV-2 and other coronaviruses

Mélade¹,

Piorkowski

Touret

et al. 2020

Preprint

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There is a need for simple reverse genetics methods to decipher the biological properties of animal and human coronaviruses. Here, we attempted to rescue the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the Feline enteric coronavirus (FeCoV) using the rapid “Infectious-Subgenomic Amplicons” (ISA) method. For each virus, transfection into permissive cells of eight overlapping subgenomic cDNA fragments covering the entire genome allowed reconstruction of the complete virus genome and generated infectious viral particles. Rescued viruses replicated the phenotypic and genotypic characteristics of the original isolates. In conclusion, the ISA method, which had been previously used for RNA viruses with shorter genomes (e.g., flaviviruses, alphaviruses and enteroviruses) can be used to rescue viruses with substantially longer genomes and usefully complements pre-existing methods for reverse genetics of coronaviruses. Its extreme simplicity and versatility makes it a strong option to decipher the biological properties of coronaviruses circulating in human, domestic or wild fauna populations.

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“…To examine the efficacy of RCA for viral rescue, we chose three cell lines for transfection: Vero, HEK293T, and BHK21 Clone 13 cells (BHK21). We selected these cell lines due to their widespread use in virus rescue [23,24]. We used a CMV-driven Mayaro virus infectious cDNA clone as both the template for our RCA reactions and as our plasmid control for the transfections (Fig.…”

Section: Peak Virus Yields Are Similar For Rca-and Plasmid-derived VImentioning

confidence: 99%

Rolling Circle Amplification is a high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones

Marano

Chuong

Weger-Lucarelli

2020

Preprint

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Alphaviruses (genus Alphavirus; family Togaviridae) are a medically relevant family of viruses that include chikungunya virus, Eastern equine encephalitis virus, and the emerging Mayaro virus. Infectious cDNA clones of these viruses are necessary molecular tools to understand viral biology and to create effective vaccines. The traditional approach to rescuing virus from an infectious cDNA clone requires propagating large amounts of plasmids in bacteria, which can result in unwanted mutations in the viral genome due to bacterial toxicity or recombination and requires specialized equipment and knowledge to propagate the bacteria. Here, we present an alternative to the bacterial-based plasmid platform that uses rolling circle amplification (RCA), an in vitro technology that amplifies plasmid DNA using only basic equipment. We demonstrate that the use of RCA to amplify plasmid DNA is comparable to the use of a midiprepped plasmid in terms of viral yield, albeit with a slight delay in virus recovery kinetics. RCA, however, has lower cost and time requirements and amplifies DNA with high fidelity and with no chance of unwanted mutations due to toxicity. We show that sequential RCA reactions do not introduce mutations into the viral genome and, thus, can replace the need for glycerol stocks or bacteria entirely. These results indicate that RCA is a viable alternative to traditional plasmid-based approaches to viral rescue. ImportanceThe development of infectious cDNA clones is critical to studying viral pathogenesis and for developing vaccines. The current method for propagating clones in bacteria is limited by the toxicity of the viral genome within the bacterial host, resulting in deleterious mutations in the viral genome, which can only be detected through whole-genome sequencing. These mutations can attenuate the virus, leading to lost time and resources and potentially confounding results.We have developed an alternative method of preparing large quantities of DNA that can be directly transfected to recover infectious virus without the need for bacteria by amplifying the infectious cDNA clone plasmid using rolling circle amplification (RCA). Our results indicate that viral rescue from an RCA product produces a viral yield equal to bacterial-derived plasmid DNA, albeit with a slight delay in replication kinetics. The RCA platform, however, is significantly more cost and time-efficient compared to traditional approaches. When the simplicity and costs of RCA are combined, we propose that a shift to an RCA platform will benefit the field of molecular virology and could have significant advantages for recombinant vaccine production.

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New reverse genetics and transfection methods to rescue arboviruses in mosquito cells

Cited by 25 publications

References 29 publications

CpG-Recoding in Zika Virus Genome Causes Host-Age-Dependent Attenuation of Infection With Protection Against Lethal Heterologous Challenge in Mice

CpG-Recoding in Zika Virus Genome Causes Host-Age-Dependent Attenuation of Infection With Protection Against Lethal Heterologous Challenge in Mice

Infectious Subgenomic Amplicons method to expedite reverse genetics of SARS-CoV-2 and other coronaviruses

Rolling Circle Amplification is a high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones

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